Prevalence and epidemiological investigation of mgrB-dependent colistin resistance in extensively drug resistant Klebsiella pneumoniae in Iran

Carbapenemases-producing K. pneumoniae are challenging antimicrobial therapy of hospitalised patients, which is further complicated by colistin resistance. The aim of this study was to investigate the molecular epidemiological insights into carbapenemases-producing and colistin-resistant clinical K. pneumoniaeA total of 162 colistin resistant clinical strains of K. pneumoniae were collected during 2017–2019. Antimicrobial susceptibility and the colistin minimum inhibitory concentration were determined. Using PCR assay, the prevalence of resistance-associated genes including blaKPC, blaIMP, blaVIM, blaOXA-48, blaNDM-1 and mcr-1 to -9 was examined. Additionally, a PCR assay was used to examine the mgrB gene in colistin-resistant bacteria. 94.4% of the tested strains were resistant to imipenem and 96.3% were resistant to meropenem. Colistin resistance (MIC > 4 µg/L) was observed in 161 isolates (99.4%) by Colistin Broth Disk Elution method. The KPC enzyme was the most common carbapenemase and was identified in 95 strains (58.6%), followed by the IMP, VIM and OXA-48 detected in 47 (29%), 23 (14.2%) and 12 (7.4%) isolates, respectively. However, no NDM-1 gene was detected. Additionally, none of the studied isolates harbored mcr variants, while mgrB gene was observed in 152 (92.6%) isolates. Colistin resistance of K. pneumoniae isolates may be associated with mgrB gene mutation. To stop the spread of resistant K. pneumoniae, surveillance must be improved, infection prevention protocols must be followed, and antibiotic stewardship must be practised.


Scientific Reports
| (2023) 13:10680 | https://doi.org/10.1038/s41598-023-37845-z www.nature.com/scientificreports/ In 2016, Yi-Yun Liu et al. discovered a plasmid-mediated colistin resistance gene (mcr-1) encoding a lipid A phosphoethanolamine transferase that confers resistance to colistin by transferring pEtN to lipid A 8 . Colistinresistant Enterobacteriaceae, particularly K. pneumoniae, that contain the mcr-1 gene have been reported from humans, animals used for food production, and the environment worldwide, raising the possibility of horizontal transmission of colistin resistance 9 . This has raised concerns about the potential emergence of pandrug resistance in Enterobacterales. As a result, it is important to continuously and precisely examine how the mcr genes emerged and propagated across bacteria. A systematic review and meta-analysis on the prevalence of colistin resistance of K. pneumoniae isolates in Iran revealed that the pooled prevalence of colistin resistance in clinical isolates was 6.9% 10 . However, the rate of K. pneumoniae carbapenem resistance was more than 73% in different studies 11,12 .
The development of reliable techniques for the detection of polymyxin resistance, with low cost and feasibility are necessary. Simner and colleagues described the Colistin Broth Disk Elution (CBDE), which uses colistin disks as a source of these antibiotics 13 . This study's objective was to examine the molecular mechanisms of colistin and carbapenem resistance among a collection of extensively drug resistant K. pneumoniae collected from clinical specimens in Tehran, Iran. Also, a comparison was made in susceptibility of K. pneumoniae isolates to colistin using Disk diffusion, Chrome Agar, E-test, and CBDE.

Materials and methods
Bacterial strains. From June 2017 to March 2019, a total of 162 non-duplicate strains of K. pneumoniae were isolated from clinical samples of inpatients and outpatients at Milad Hospital in Tehran, and they were resistant to the colistin in the initial screening. The disc diffusion method and selective agar medium CHRO-Magar COL-APSE (Paris, France) were used to detect resistance to colistin and finally colistin resistant isolates that were confirmed by both methods were included in the study.
Molecular analysis of carbapenem and colistin resistance-associated genes. DNA extraction was performed commercial High pure PCR template preparation kit (Roche Molecular Biochemicals, IN, USA) according to the manufacturer's instructions. Carbapenemases-encoding genes such as (blaKPC, blaIMP, blaVIM, blaOXA-48 and blaNDM-1) were detected by singleplex PCR [15][16][17][18][19] . Singleplex PCRs were also used to test the colistin-resistant isolates for the presence of mcr-1-9 according to previous published papers [20][21][22][23][24] . In addition, using specific primers that targeted the mgrB coding sequence and several flanking areas, PCR analysis of mgrB was carried out 25 . The sequence of primers used in this study, which were selected based on common variants, is shown in Table 1. Positive strains for blaNDM, blaVIM, blaKPC and blaOXA-48 that confirmed by sequencing method were used as positive control and E. coli ATCC 25922 was utilized as negative control in the PCR assays.
Genomic sequencing. Draft genome sequences were created using genomic sequencing on an Oxford Nanopore GridION in National Reference Laboratory. Statistical analysis. Data analysis was performed using IBM SPSS Statistics v.22.0 (IBM Corp., Armonk, NY). The frequency of isolates between groups was compared by χ2 test. A P-value of ≤ 0.05 was considered statistically significant.

Results
Epidemiology of colistin-resistant K. pneumoniae isolates. During the study periods, 162 colistin resistant strains of K. pneumoniae belonging to 94 (58%) men and 68 (42%) women were studied. The average age of the patients was 67.25 ± 1.2 (between 19 and 98 years) and most of the studied patients 103 (63.6%) were older than 65 years. Out of 162 patients, 155 were inpatients and only 7 were outpatients. The largest number of studied samples included trachea (41,25.3%), urine (37,22.8%) and sputum (35,21.6%) samples, more than half of which (95, 58.6%) were received from the intensive care unit (ICU). The demographic characteristics of patients, sample types and distribution of study isolates among the different hospital wards are shown in Table 2.
Antimicrobial susceptibility testing. 79.6% of the tested strains were resistant to cotrimoxazole, and except for amikacin, resistance to other antibiotics was observed above 90% ( Fig. 1). Out of 162 K. pneumoniae isolates, 161 (99.3%) were resistant to ciprofloxacin. The resistance rate of the third generation of cephalosporins including ceftriaxone and ceftazidime was 98.8%. Resistance to carbapenems, which are used as alternative antibiotics in the treatment of strains resistant to cephalosporins, was high, so that 94.4% of the tested strains were resistant to imipenem and 96.3% were resistant to meropenem. From aminoglycoside antibiotics, gentamycin and amikacin were tested, and the resistance to gentamycin was about 90%, while the resistance to amikacin Molecular analysis of carbapenem and colistin resistance-associated genes. The gene encoding the KPC enzyme was the most common and was identified in 95 strains (58.6%), followed by IMP and VIM in 47 (29%) and 23 (14.2%) strains, respectively. The gene encoding the OXA-48 enzyme was found in 12 strains (7.4%), while the NDM-1 gene was not detected. PCR screening for plasmid-mediated colistin resistance genes (mcr-1 to mcr-9) were examined on tested strains. According to the findings, no positive mcr isolates were found. Therefore, all the strains were examined for the presence of the mgrB gene, and 152 (92.6%) strains harboured the gene. Genomic sequencing of strains harboured mgrB gene revealed that only the conversion of codon 39 from TCA to TGA was present in two isolates, since the TGA codon is a stop codon, which causes the termination of transcription and the loss of protein function (Accession number KF852760: m colistin-susceptible K. pneumoniae strain; coverage:53%).

Discussion
The global increase in multidrug-resistant K. pneumoniae strains has increased the use of colistin to treat these infections, resulting in the emergence of colistin resistance worldwide 26,27 . The important concern that must be considered in the management of nosocomial infections caused by K. pneumoniae are periodic surveillance to identify the resistant strains for optimizing available infection control policies and treatment options in different parts of the hospitals 28 . This study investigated the molecular mechanisms of colistin and carbapenem resistance among a collection of extensively drug resistant K. pneumoniae clinical isolates in Tehran, Iran. Also, different phenotypic methods including disc diffusion, E-test, chrome agar and CBDE were compared to determine the susceptibility of K. pneumoniae isolates to colistin.
In the comparison between the phenotypic methods, the highest number of colistin-resistant strains was identified with the CBDE method. So that out of 162 tested strains, except for one strain, the other isolates were resistant to colistin by the CBDE method, then the E-test and chrome agar methods showed the highest resistance (95.6% overlap to detect colistin resistance), and finally, the disc diffusion method showed the lowest resistance. Some studies have compared agar dilution, E-test and disk diffusion methods to measure colistin resistance in Table 1. Primers used in this study. KPC  ATG TCA CTG TAT CGC CGT CT  TTT TCA GAG CCT TAC TGC CC  893   15   NDM-1  GGT TTG GCG ATC TGG TTT TC  CGG AAT GGC TCA TCA CGA TC  621   16   OXA-48  TTG GTG GCA TCG ATT ATC GG  GAG CAC TTC TTT TGT GAT GGC  743   17   IMP  CCA AAC YACTASGTT ATC  GAA TAG RRT GGC TTA AYT CTC  188     www.nature.com/scientificreports/ Gram-negative bacilli 29,30 . According to CLSI and EUCAST guidelines, they confirmed that the use of diffusionbased methods for antimicrobial susceptibility testing against colistin was unreliable, which is due to the large size of the colistin, prevents its uniform diffusion in agar-containing media 14 . Despite this recommendation, the results of this study showed that the disk diffusion method performed with both types of commercial disks containing colistin was able to successfully differentiate resistant strains. According to this result, it seems that revisions should be made about the diagnostic value of the disc diffusion method to determine the susceptibility to colistin and this test should not be completely abandoned. Because disc diffusion method is able to identify at least strains with high level of resistance to colistin and can be an important tool in the direction of rapid screening of resistant strains with high resistance level. CBDE is a simple and low-cost phenotypic method to test antimicrobial sensitivity against colistin in Gram-negative bacilli, including Enterobacteriaceae, which has been approved by CLSI and EUCAST. Studies have shown that CBDE method was comparable with the broth microdilution method as a reference method with a 100% correlation 13,31 .

Primers Sequences Size (bp) References
The colistin-resistant rate in Iran were reported about 11.6% 32 . Nevertheless, data from reports of neighboring countries showed the resistance to colistin are ranging from 0 to 31.7% 33 . As expected for colistin-resistant isolates in our study, most were also resistant to other clinically useful antimicrobial agents. In the present study, 96% of isolates showed phenotypic resistance to meropenem and/or imipenem, which similar to many other studies, 95 of them harboured bla KPC gene 34 . We found that none of the colistin-resistant K. pneumoniae isolates had plasmid-encoded mcr-genes, suggesting that the resistance is mediated by chromosomally encoded mechanisms. MCR-1 is still extremely uncommon in clinical isolates worldwide 35 . In previous investigations, mcr-1 prevalence in Enterobacteriaceae was reported to range between 0.1 and 1% 36,37 . The literature shows the prevalence of the mcr-1 gene was lower in Enterobacteriaceae strains isolated from human sources than in strains isolated from animal and food samples 38 . This assumes that their reservoir is at least in animals and the environment, following the important use of colistin in animal production and in general agriculture. In Iran, the massive use of colistin in clinical practice following the spread of carbapenemase-producing Enterobacteriaceae has led to the selection of multidrug-resistant bacteria in hospital settings 39 .
Interestingly, the results of the current study showed that mcr-1-negative K. pneumoniae isolates had high level of colistin resistance. Consistent with this findings, previous studies reported that K. pneumoniae with chromosomal mutations in mgrB exhibited a high level of colistin resistance 40,41 . The mechanisms of colistin resistance other than those attributed to the mcr-1 gene in the K. pneumoniae isolates in this study are being further studied to define the precise molecular mechanism of resistance. Of 162 colistin resistant K. pneumoniae isolates, 150 (92.6%) isolates had mgrB. Critical changes in mgrB, such as disruption of the promoter or coding sequence, are thought to cause the gene to be silenced or lead to the generation of shortened mgrB 40 . In reality, PhoP/PhoQ activation follows mgrB inactivation by any of these occurrences, which in turn activates the PmrA response regulator responsible for modification of the lipopolysaccharide polymyxin target 25 . So mentioned mutation in the present study may causes the termination of transcription and the loss of protein function. Avgoulea et al. reported that insertional inactivation of the mgrB gene conferred resistance to colistin in all isolates tested 42 .
There were some limitations related to the present study. Firstly, it should be noted that this study was performed using extracted DNA samples from K. pneumoniae that were selected based on the results of initial screening for colistin resistance. The lack of strains carrying mcr genes except mcr-1 as a positive control is another limitation. From an epidemiological standpoint, the monocentric nature of the study is a significant limitation.

Conclusion
Our findings indicate that the colistin resistance of K. pneumoniae isolates may be associated with mgrB gene mutation. These data provide added insight into the mechanism of colistin. Colistin resistance developed with a number of new mutations among highly resistant populations, limiting the availability of further antimicrobial medicines and resulting in pandrug resistance. The prevalence of resistance to carbapenems and colistin in Iran should be surveyed and new therapeutic strategies including old drugs should be evaluated and used in Iran.

Data availability
All data generated or analyzed during this study are included in this published article and Supplementary Information file [and its tables and figures].

Code availability
The code is available from the corresponding author upon request.